During the aging process, the number and multipotentiality of quiescent stem cell populations and their representation in developed tissues diminishes (Sommer et al., 2002, Prog. Neurobiol. 66:1-18). A major goal of modem biology is to understand the molecular mechanisms that underlie stem cell self-renewal in hopes of either facilitating the long-term expansion of adult stem cell populations ex vivo without a loss of phenotypic multipotentiality for either therapeutic transplantation strategies or activation and the expansion of stem cell populations to modulate tissue homeostasis in vivo. Attempts to experimentally define molecular phenotypes underlying “stemness” have applied cell and genetic approaches to identify several genes that may affect the self-renewal process including Nanog (Cavaleri et al., 2003, Cell 113:551-552; Chambers et al., 2003, Cell 113:643-655; Hirata et al., 2001, EMBO J. 20:4454-4466; Mitsui et al., 2003, Cell 113:631-642), Nucleostemin (Tsai et al., 2002, Genes Dev. 16:2991-3003), Oct4 (Chambers et al., 2003, Cell 113:643-655; Pan et al., 2002, Cell Res. 12:321-329) and Bmi1 (Park et al., 2004, J. Clin. Invest. 113:175-179). However, the mechanisms that underlie stem cell self-renewal remain to be determined.
Notch signaling plays a key role in normal development through diverse effects on differentiation, survival, and proliferation and these events are highly dependent on signal strength and cellular context (Artavanis-Tsakonas et al., 1995, Science 268:225-232; Kadesch, T., 2000, Exp. Cell. Res. 260:1-8). In the canonical pathway, four Notch transmembrane receptor genes, Notch1-4 (Kadesch, T., 2000, Exp. Cell. Res. 260:1-8), and three transmembrane ligand gene families: Jaggedl-3 (Lindsell et al., 1995, Cell 80:909-917; Shawber et al., 1996, Dev. Biol. 180:370-376), Delta1-4 (Bettenhausen et al., 1995, Development 121:2407-2418; Dunwoodie et al., 1997, Development 124:3065-3076; Shutter et al., 2000, Genes Dev. 14:1313-1318) and F3/Contactin (Hu et al., 2003, Cell 115:163-175) have been described to date (Mumm et al., 2000, Dev. Biol. 228:151-165), and these transmembrane ligands activate Notch receptors on neighboring cells (Artavanis-Tsakonas et al., 1995, Science 268:225-232; Iso et al., 2003, Arterioscler. Thromb. Vasc. Biol. 23:543-553; Kimble et al., 1997, Annu. Rev. Cell Dev. Biol. 13:333-361). Notch signaling involves the proteolytic cleavage of Notch which generates an intracellular domain that is able to translocate to the nucleus (Baron, M., 2003, Sernin. Cell Dev. Biol. 14:113-119; Radtke et al., 2003, Nat. Rev. Cancer. 3:756-767) and activate CSL1 (for CBF-1/RBPJk/KBF2 in mammals; Su(H) in Drosophila and Xenopus; and Lag-2 in C. elegans) family of transcription factors-dependent transcription (Lai, 2002, EMBO Rep. 3:840-845). In contrast, soluble forms of functional Notch ligands that contain only the extracellular domain have also been described (Hicks et al., 2002, J. Neurosci. Res. 68: 655-667; Qi et al., 1999, Science 283:91-94; Zimrin et al., 1996, J. Biol. Chem. 271:32499-32502). Notch receptors have also been reported to regulate cellular processes through CSL-independent pathways that may involve interactions with other signaling molecules such as NF-κB and Src (Nofziger et al., 1999, Development 126: 1689-1702; Small et al., 2001, J. Biol. Chem. 276:32022-32030; Shawber et al. 1996, Development 122: 3765-3773). Phenotypic analysis of mice null for Notch receptors or their ligands emphasizes the requirement for proper Notch signaling not only during development but in the adult as well (Conlon et al. 1995, Development 121: 1533-1545; Hamada et al., 1999, Development 126: 3415-3424; Jiang et al., 1998, Genes Dev. 12: 1046-1057; Krebs et al., 2000, Genes Dev. 14: 1343-1352; Xue et al., 1999 Hum. Mol. Genet. 8: 723-730). Indeed, aberrant Notch signaling has been implicated in several human pathological conditions including the development of the CADASIL (Joutel et al. 1996, Nature 383: 707-710) and Alagille syndromes (Li et al., 1997, Nat. Genet. 16: 243-251; Li et al., 1997, Nat. Genet. 16: 243-251) and the formation of neoplasias in mice and humans (Rae et al., 2000, Int. J. Cancer 88: 726-732; Rohn et al. 1996, J. Virol. 70: 8071-8080; Zagouras et al., 1995, Proc. Natl. Acad. Sci. USA 92: 6414-6418).
Because perturbations in the regulation of Notch signaling have been implicated in malignant transformation (Maillard et al., 2003, Immunity 19:781-791; Radtke et al., 2003, Nat. Rev. Cancer. 3:756-767) and tumor suppression function (Nicolas et al., 2003, Nat. Genet. 33:416-421; Radtke et al., 2003, Nat. Rev. Cancer. 3:756-767), similar Notch signals may potentially contribute to the expansion of undifferentiated stem cells (Hitoshi et al., 2002, Genes Dev. 16:846-858; Nickoloff et al., 2003, Oncogene 22:6598-6608; Tropepe et al., 2001, Neuron 30:65-78).
The regulation of Notch signaling is vital in the genesis, ontogeny and lineage specification of neural stem cell (NSC) populations (Morrison et al., 2001, Curr. Opin. Cell. Biol. 13:666-672). In the central nervous system, a two-step requirement for Notch signaling in gliogenic differentiation has been proposed (Grandbarbe et al., 2003, Development 130:1391-1402), and these events are required for the maintenance but not formation of neural stem cells (Hitoshi et al., 2002, Genes Dev. 16:846-858). Indeed, populations of NSC have been generated independent of Notch signaling using embryonic stem cells from null mice (Hitoshi et al., 2002, Genes Dev. 16:846-858), and these cells are quickly depleted in the early embryonic brains of mice with diminished Notch signaling capacity as exhibited by RBP-Jkappa(−/−), Notch1(−/−) and presenillin1 (−/−) mice (Hitoshi et al., 2002, Genes Dev. 16:846-858). However, later in development, Notch signaling is required for astrocytic differentiation of NSC populations (Tanigaki et al., 2001, Neuron 29:45-55). In the peripheral nervous system, the addition of a soluble non-transmembrane form of the Notch ligand, Delta1, to clonally-derived populations of neural crest stem cells (NCSC) leads to an immediate and irreversible loss of neurogenic potential including differentiation into glia (Morrison et al., 2001, Curr. Opin. Cell. Biol. 13:666-672). Interestingly, this response can also be reproduced in NCSC populations by the expression of a constitutively active form of Notch1 (Morrison et al., 2001, Curr. Opin. Cell. Biol. 13:666-672). Therefore, numerous gain-of-function Notch signaling events may contribute to instructive gliogenic NSC differentiation (Wang et al., 2000, Neuron 27:197-200), but the consequences of suppressing endogenous Notch signaling in these oells have not been studied.
Neurological damage in a mammal having as its genesis trauma, tumor formation or a genetic or other component, is very difficult to treat and/or reverse in the mammal. One treatment for neurological damage to the central nervous system is neurotransplantation. Over the last few decades, neurotransplantation has been used to explore the development, plasticity, and regeneration of the central nervous system (McKay, 1997, Science 276:66-71). Also, neurotransplantation has been used to effect the repair and functional restoration of diseased and damaged nervous tissues (Bjorklund, 1993, Nature 362:414-415; Olson, 1997, Nature Med. 3:1329-1335; Spencer et al., 1992, N. Engl. J. Med. 327:1541-1548: Freed et al., 1992, N. Engl. J. Med. 327:1549-1555; Kordower et al., 1995, N. Engl. J. Med. 332:1118-1124; Defer et al., 1996, Brain 119:41-50; Lopez-Lozano et al., 1997, Transp. Proc. 29:977-980; Rosenstein, 1995, Exp. Neurol. 33:106; Turner et al., 1993, Neurosurg. 33:1031-1037; Kang et al., 1993, J. Neurosci. 13:5203-5211; Andersson et al., 1993, Int. J. Dev. Neurosci. 11:555-568; Sanberg et al., 1997, Nature Med. 3:1129-1132).
In particular, a series of human patients with Parkinson's disease have been treated by neurotransplantation of mesencephalic cells obtained from 6 to 9 week old abortuses of human fetuses (Spencer et al., 1992, N. Engl. J. Med. 327:1541-1548: Freed et al., 1992, N. Engl. J. Med. 327:1549-1555; Kordower et al., 1995, N. Engl. J. Med. 332:1118-1124; Defer et al., 1996, Brain 119:41-50; Lopez-Lozano et al., 1997, Transp. Proc. 29:977-980). Some of the patients exhibited significant improvement both in clinical symptoms and in the synthesis of dopamine, as assessed by fluorodopa uptake using positron-emission tomography (Spencer et al., 1992, N. Engl. J. Med. 327:1541-1548: Freed et al., 1992, N. Engl. J. Med. 327:1549-1555; Kordower et al., 1995, N. Engl. J. Med. 332:1118-1124; Defer et al., 1996, Brain 119:41-50). However, the process of obtaining fetal tissue for therapeutic uses has presented major logistic and ethical barriers (Rosenstein, 1995, Exp. Neurol. 33:106; Turner et al., 1993, Neurosurg. 33:1031-1037). Also, only about 5 to 10% of dopaminergic neurons survive, apparently because of adverse immune reaction to the same (Lopez-Lozano et al., 1997, Transp. Proc. 29:977-980) and because the fetal tissue is primarily dependent on lipid instead glycolytic metabolism (Rosenstein, 1995, Exp. Neurol. 33:106). For these reasons, attempts have been made to develop alternative cells such as fibroblasts (Kang et al., 1993, J. Neurosci. 13:5203-5211), fetal astrocytes (Andersson et al., 1993, Int. J. Dev. Neurosci. 11:555-568), and sertoli cells (Sanberg et al., 1997, Nature Med. 3:1129-1132) which are suitable for neurotransplantation.
In order to treat diseases, disorders, or conditions of the central nervous system, such as for example brain tumors, brain trauma, Huntington's disease, Alzheimer's disease, Parkinson's disease, and spinal cord injury, by transplantation, donor cells should be easily available, capable of rapid expansion in culture, immunologically inert, capable of long term survival and integration in the host brain tissue, and amenable to stable transfection and long-term expression of exogenous genes (Bjorklund, 1993, Nature 362:414-415; Olson, 1997, Nature Med. 3:1329-1335). Thus, there is a long-felt and acute need for methods for clonally expanding a stem cell, and stem cells produced thereby, for use in regenerative medicine, or therapeutic cloning. Therefore, there is a long-felt need for methods to create pluripotent stem cells that are genetically matched to a patient in order to generate personalized tissues that would combat the ravages of aging and disease without organ rejection or other complications that plague conventional transplant therapy.
The advantage of using autologous adult stem cells for regenerative medicine lies in the fact that they are derived from and returned to the same patient, and are therefore not subject to immune-mediated rejection. The major drawback is that these cells lack the plasticity and pluripotency of ES cells and thus their potential is uncertain. To overcome this hurdle, much attention has been directed towards bone marrow cells, which can differentiate into such diverse tissues as bone, cartilage, and muscle (Pittenger et al., 1999, Science 284:143-147). It has also been shown that mouse bone marrow cells, when injected directly into infracted mouse hearts, can develop into myocytes and vascular structures (Orlic et al., 2001, Nature 410:701-705). Similar experiments have shown that mouse bone marrow cells delivered intravascularly are able to migrate to the central nervous system and eventually exhibit neuron-like phenotypes (Brazelton et al., 2000, Science 290:1775-1779; Mezey et al., 2000, Science 290:1779-1782).
The regenerative capacity of adult cells is not limited to those stem cells obtained derived from bone marrow. That is, neural stem cells are capable of differentiating into blood cells when transplanted into bone marrow (Bjornson et al., 1999, Science 283:534-537). However, although these results are promising, the number of pluripotent bone marrow and neural stem cells is limited. Although the ability to produce large number of pluripotent stem cells would have immediate clinical applications, such as the generation of lines from patients (e.g., cancer, cardiovascular diseases, and neurodegenerative diseases) which would provide cell-based therapy to cure these diseases, the clonal expansion of these cells has not been possible. Thus, there is an acute need for methods for clonally expanding pluripotent human stem cells without loss of pluripotency, and cells produced thereby for use in numerous therapies, and the present invention meets these needs.
The functional integrity of the human vascular system is maintained by the endothelial cell which monitors the non-thrombogenic interface between blood and tissue in vivo. Thus, factors that influence human endothelial cell function may contribute significantly to the regulation and maintenance of homeostasis (see Maciag, 1984, In: Progress in Hemostasis and Thrombosis, pp. 167-182, Spaet, ed., A. R. Liss, New York; Folkman and Klagsburn, 1987, Science 235:442-447; Burgess and Maciag, 1989, Annu. Rev. Biochem. 58:575-606). Likewise, events that perturb this complex equilibrium are relevant to the pathophysiology of human disease states in which cellular components of the vascular tree are active participants including, e.g., atherogenesis, coronary insufficiency, hypertension, rheumatoid arthritis, solid tumor growth and metastasis, and wound repair.
Since the endothelium is present in all organs and tissues, endothelial cell function is also fundamental to the physiology and integration of these multicellular systems. This includes the ability to monitor and interface with repair systems that employ the tightly regulated inflammatory, angiogenic and neurotropic responses. Indeed, biochemical signals that are responsible for the modification of these responses have been well characterized as polypeptide growth factors and cytokines; however, their mechanisms of operation have, prior to the present invention, been poorly understood, impeding their acceptance as valuable tools in clinical management.
During the past decade, differential cDNA cloning methods, including e.g., conventional subtractive hybridization (Hla and Maciag, 1990, Biochem. Biophys. Res. Commun. 167:637-643), differential polymerase chain reaction (PCR)-oriented hybridization (Hla and Maciag, 1990, J. Biol. Chem. 265:9308-9313), and more recently, a modification of the differential display (Zimrin et al., 1995, Biochem. Biophys. Res. Commun. 213:630-638) were used to identify genes induced during the process of human umbilical vein endothelial cell (HUVEC) differentiation in vitro. Very early studies disclosed that HUVEC populations are able to generate capillary-like, lumen-containing structures when introduced into a growth-limited environment in vitro (Maciag et al., 1982, J. Cell Biol. 94:511-520). These studies permitted the identification and characterization of protein components of the extracellular matrix as inducers of this differentiation process, while at the same time defining the capillary-like structures as non-terminally differentiated (Maciag, 1984, In: Progress in Hemostasis and Thrombosis, pp. 167-182, Spaet, ed., A. R. Liss, New York). Additional experiments have elucidated the importance of polypeptide cytokines, such as IL-1 (Maier et al., 1990, J. Biol. Chem. 265:10805-10808) and IFNγ (Friesel et al., 1987, J. Cell Biol. 104:689-696), as inducers of HUVEC differentiation in vitro, and ultimately lead to an understanding that the precursor form of IL-1α was responsible for the induction of HUVEC senescence in vitro (Maciag et al., 1981, J. Cell Biol. 91:420-426; Maier et al., 1990, Science 249:1570-1574).
Recent research has employed differential cDNA cloning methods, which permits the identification of new and very interesting genes. However, until very recently, establishing their identity did not provide insight into the mechanism of HUVEC differentiation. Current research has focused upon the fibroblast growth factor (FGF) and interleukin (IL)-1 gene families as regulators of the angiogenesis process, both in vitro and in vivo (Friesel et al., 1995, FASEB J. 9:919-925; Zimrin et al., 1996, J. Clin. Invest. 97:1359). The human umbilical vein endothelial cell (HUVEC) has proven to be an effective model for studying the signal pathways utilized by FGF-1 to initiate HUVEC migration and growth, the role of IL-1α as an intracellular inhibitor of FGF-1 function and modifier of HUVEC senescence, and the interplay between the FGF and the IL-1 gene families as key effectors of HUVEC differentiation in vitro. Such insight has enabled the present inventors to use modem molecular methods to identify a key regulatory ligand-receptor signaling system, which is able to both induce capillary endothelial cell migration and repress large vessel endothelial cell migration.
Because members of the fibroblast growth factor (FGF) family are also known as potent regulators of developmental, physiological and pathophysiologic events in mammals, the FGF family has been the subject of numerous comprehensive reviews related to the field of cellular differentiation. (See, e.g., Burgess and Maciag, 1989, Annu. Rev. Biochem. 58: 575-606; Friesel and Maciag, 1995, FASEB J. 9: 919-925; Rifkin and Moscatelli, 1989, J. Cell Biol. 109: 1-6; Klagsbrun and Baird, 1991, Cell 67: 229-231. The best characterized members of the FGF family are FGF-1 (acidic FGF) and FGF-2 (basic FGF). These two proteins are unusual growth factors in that they lack a classical signal sequence to direct their secretion through the conventional endoplasmic reticulum (ER)-Golgi apparatus. Ligation of a signal sequence to the FGF prototypes yields functional extracellular ligands with potent transforming potential in vitro and the ability to induce exaggerated angiogenic and neurotropic phenomena in vivo including the generation of atheroma-like lesions.
Because the mitogenic activities of FGF-1 and FGF-2 are mediated by a high affinity receptor at the plasma membrane surface, it has been proposed that a tightly regulated, yet unconventional, export pathway exists to regulate the export of these growth factors. Consequently, the regulation of human endothelial cell growth and the role of FGF-1 in mediating this process in vivo has been the subject of a number of studies. See, e.g., Maciag et al., 1979, Proc. Natl. Acad. Sci. USA 76: 5674-5678; Maciag et al., 1981, J. Cell Biol. 91: 420-426; Maciag et al., 1982, J. Cell Biol. 94: 511-520; Maciag et al., 1984, Science 225: 932-935).
The addition of a signal sequence (ss) to either FGF-1 or FGF-2 established the function of genes encoding these modified FGF proteins as transforming genes in a variety of target cells. See, e.g., Rogelj et al., 1988, Nature 331: 173-175; Blam et al., Oncogene. 3: 129-136); Jouanneau et al., 1991, Proc. Natl. Acad. Sci. USA 88: 2893-2897; Talarico and Basilico, 1991, Mol. Cell. Biol. 11:1138-1145; Forough et al., 1993, J. Biol. Chem. 268: 2960-2968). In vivo expression studies in the porcine iliac artery using a liposome:vector gene transfer method demonstrate an exaggerated hypertrophic response to the presence of extracellular FGF-1 including the formation of a prominent neointima containing numerous capillary and aorta-like structures (Nabel et al., 1993, Nature 362: 844-846). In a similar manner, a transgene expressing a FGF-44(ss):FGF-1 chimera under control of the α-crystallin promoter, which is active during lens development in the eye, induces a hypertrophic response that includes inappropriate formation of blood vessels and nerve bundles in the lens (Overbeek et al., 1994, Development).
In both studies, the transfer of the wild-type FGF-1 cDNA failed to yield pathologic consequences. Prior studies (e.g., Thompson et al., 1988, Science 241: 1349-1352) have suggested that FGF-1 may have evolved without a functional signal sequence because expression would be accompanied by inappropriate export during certain situations, resulting in the formation of exaggerated vascular and neuronal structures. Thus, it appears that the pathway for FGF-1 export is tightly regulated in order to control the angiogenic and neurogenic activities of the protein.
Thrombin is also known to play a role in angiogenesis, as well as modulation of cell growth and differentiation. Thrombin is able to elicit many cellular responses, including those that are thrombotic, inflammatory, proliferative and atherosclerotic. Such thrombin-mediated cellular responses are mediated by proteolytic activation of a specific cell surface receptor known as the thrombin receptor, which is a tethered ligand receptor (Vu et al., 1991, Cell 64: 1057-1068; Rasmussen et al., 1991, FEBS Lett. 288: 123-128; Zhong et al., 1992, J. Biol. Chem. 267: 16975-16979; Bahou et al., 1993, J. Clin. Invest. 91: 1405-1413; McNamara et al., 1993, J. Clin. Invest. 91: 94-98; Glembotski, 1993, J. Biol. Chem. 268: 20646-20652; and Park et al., 1994, Cardiovasc. Res. 28: 1263-1268). The thrombin receptor has seven transmembrane-spanning domains and belongs to the family of G-protein coupled receptors (Vu et al., 1991, Cell 64: 1057-1068 and Schwartz, 1994, Current Opin. Biotechnol. 5: 434-444). Activation of the receptor occurs by thrombin cleavage of an extracellular N-terminal domain. The new N-terminus—through intramolecular interaction—activates the receptor (Vu et al., 1991, Cell 64: 1057-1068; Coughlin, 1993, Thromb. Haemostas. 70: 184-187; Van Obberghen-Schilling and Pouyssegur, 1993, Thromb. Haemostas. 70: 163-167; Brass et al., 1994, Ann. NY Acad. Sci. 714: 1-12).
Thrombin is also known to have a variety of activities in different cell types and thrombin receptors are known to be present in such cell types as human platelets, vascular smooth muscle cells, endothelial cells and fibroblasts. For example, thrombin is important in periodontal wound healing and associated inflammatory processes by promoting the growth and contraction of gingival fibroblasts (Chang et al., 2001, J. Periodontol. 72:303-13). Such periodontal stimulatory effects of thrombin are associated with its protease activation of thrombin receptors. It follows that substances with the ability to mediate the function of thrombin may be useful in the treatment of thrombotic, inflammatory, atherosclerotic and fibroproliferative disorders, as well as other disorders in which thrombin and its receptor play a pathological role.
In addition to the thrombin and FGF-1 signaling systems, the Jagged/Serrate/Delta-Notch/Lin/Glp signaling system, originally described during the development of C. elegans and Drosophila, is a cell signaling system with a critical role in cellular growth and differentiation. The Jagged/Serrate/Delta-Notch/Lin/Glp signaling system, instrumental in cell fate decisions, has been found to be highly conserved in mammalian cells (Nye and Kopan, 1995, Curr. Biol. 5:966-969). Notch proteins comprise a family of closely-related transmembrane receptors initially identified in embryologic studies in Drosophila (Fortini and Artavanis-Tsakonas, 1993, Cell 75:1245-1247). The genes encoding the Notch receptor show a high degree of structural conservation, and contain multiple EGF repeats in their extracellular domains (Coffinan et al., 1990, Science 249:1438-1441; Ellisen et al., 1991, Cell 66:649-661; Weinmaster et al., 1991, Development 113:199-205; Weinmaster et al., 1992, Development 116:931-941; Franco del Amo et al., 1992, Development 115:737-744; Reaume et al., 1992, Dev. Biol. 154:377-387; Lardelli and Lendahi, 1993, Mech. Dev. 46:123-136; Bierkamp and Campos-Ortega, 1993, Mech. Dev. 43:87-100; Lardelli et al., 1994, Exp. Cell Res. 204:364-372). In addition to the thirty-six EGF repeats within the extracellular domain of Notch 1, there is a cys-rich domain composed of three Notch Lin Glp (NLG) repeats, which is important for ligand function, followed by a cys-poor region between the transmembrane and NLG domain.
The intracellular domain of Notch 1 contains six ankyrin/Cdc10 repeats positioned between two nuclear localization sequences (NLS) (Artavanis-Tsakonas et al., 1995, Science 268:225-232). This motif is found in many functionally diverse proteins (see, e.g., Bork, 1993, Proteins 17:363-374), including members of the Rel/NF-κB family (Blank et al., 1992, TIBS 17:135-140), and is thought to be responsible for protein-protein interactions. Notch has been shown to interact with a novel ubiquitously distributed cytoplasmic protein deltex through its ankyrin repeats, a domain shown by deletion analysis to be necessary for activity (Matsuno et al., 1995, Development 121:2633-2644).
Several Notch ligands have been identified in vertebrates, including Delta, Serrate and Jagged. The Notch ligands are also transmembrane proteins, having highly conserved structures. These ligands are known to signal cell fate and pattern formation decisions through the binding to the Lin-12/Notch family of transmembrane receptors (Muskavitch and Hoffmann, 1990, Curr. Top. Dev. Biol. 24:289-328; Artavanis-Tsakonas and Simpson, 1991, Trends Genet. 7:403-408; Greenwald and Rubin, 1992, Cell 68:271-281; Gurdon, 1992, Cell 68:185-199; Fortini and Artavanis-Tsakonas, 1993, Cell 75:1245-1247; and Weintraub, 1993, Cell 75:1241-1244). A related protein, the Suppressor of hairless (Su(H)), when co-expressed with Notch in Drosophila cells, is sequestered in the cytosol, but is translocated to the nucleus when Notch binds to its ligand Delta (Fortini and Artavanis-Tsakonas, 1993, Cell 75:1245-1247). Studies with constitutively activated Notch proteins missing their extracellular domains have shown that activated Notch suppresses neurogenic and mesodermal differentiation (Coffinan et al., 1993, Cell 73:659-671; Nye et al., 1994, Development 120:2421-2430).
The Notch signaling pathway, which is activated by Jagged1 in the endothelial cell, involves cleavage of the intracellular domain by a protease, followed by nuclear trafficking of the Notch fragment and the interaction of this fragment with the KBF2/RBP-Jk transcription factor (Jarriault et al., 1995, Nature 377:355-358; Kopan et al., 1996, Proc. Natl. Acad. Sci. USA 93:1683-1688), a homolog of the Drosophila Suppressor of hairless gene (Schweisguth et al., 1992, Cell 69:1199-1212), a basic helix-loop-helix transcription factor involved in Notch signaling in insects (Jennings et al., 1994, Development 120:3537-3548) and in the mouse (Sasai et al., 1992, Genes Dev. 6:2620-2634). This effector is able to repress the transcriptional activity of other genes encoding transcription factors responsible for entry into the terminal differentiation program (Nye et al., 1994; Kopan et al., 1994, J. Cell. Physiol. 125:1-9).
The Jagged1 gene encodes a transmembrane protein which is directed to the cell surface by the presence of a signal peptide sequence (Lindsell et al., 1995, Cell 80:909-917). While the intracellular domain contains a sequence with no known homology to intracellular regions of other transmembrane structures, the extracellular region of the ligand contains a cys-rich region, 16 epidermal growth factor (EGF) repeats, and a DSL (Delta Serrate Lag) domain. The DSL domain as well as the EGF repeats, are found in other genes including the Drosophila ligands, Serrate (Baker et al., 1990, Science 250:1370-1377; Thomas et al., 1991, Development 111:749-761) and Delta (Kopczynski et al., 1988, Genes Dev. 2:1723-1735), and C. elegans genes Apx-1 (Henderson et al., 1994, Development 120:2913-2924; Mello et al., 1994, Cell 77:95-106) and Lag-2 (Tax et al., 1994, Nature 368:150-154).
Until the discovery of the invention disclosed in International Application WO 97/45143 (the disclosure of which is incorporated by reference herein in its entirety), human Jagged1 remained undefined and the function and relationship, if any, of the human ligand to Notch remained unknown in the art. The human Jagged1 gene and soluble forms thereof have now been cloned, isolated and defined, and the Jagged1-Notch role in endothelial cell differentiation and/or migration has been elucidated. The novel Jagged1-Notch signaling pathway produces disparate effects on the migration of large and small vessel endothelial cells, providing what appears to be the first demonstration of a signaling difference between large and small vessel endothelial cells both in degree and direction. This highlights the potential function of a previously unknown ligand-receptor signaling pathway in the endothelial cell which is modulated during the migratory phase of angiogenesis. WO 97/45143 also discloses the previously unresolved phenomenon in which endothelial cells have been shown to reproducibly differentiate into a non-terminal and completely reversible tubular-like cell phenotype in vitro (Maciag et al., 1982, J. Cell Biol. 94:511-520). Further, the WO 97/45143 also discloses a method for preventing the undesirable migration of specific cell types into large blood vessels following angioplastic surgery to control restenosis.
It is desirable to identify the bases of cellular differentiation, with the ultimate goal of gaining the understanding and ability to provide targeted, precise therapy to patients in need of therapy for differentiation-related disorders, such as those affecting inflammatory responses, atherogenic responses, and neurotropic responses. For example, in view detrimental effects that may ensue as a result of FGF-1 export, effective regulation is needed. International Patent Publication No. WO 03/018595A2 of Maciag et al. (incorporated by reference herein in its entirety) discloses that FGF-1 is exported in response to various stimuli, including stressors (such as temperature stress), as an FGF-1 complex comprising, inter alia, p40 Syn1 and S100A13. Formation of the FGF-1 complex may also be cofactor-mediated, and complexation of FGF-1 with Syn-1 may occur under “non-stress” conditions as well. Further, complexes including FGF-1 and Syn-1 may be involved in processes of FGF-1 export and regulation.
Characterization of FGF-1 as a potent promoter of angiogenesis when elaborated into the extracellular milieu in vivo links FGF-1 to physiologic regulation of FGF-1 export (see, e.g., Folkman et al., 1987, Science 235:442-447) associated with numerous disease processes. There is a long-term unfulfilled need to develop therapeutics for such disease processes as inflammation, thrombosis, and to regulate angiogenesis, and to provide treatment, and even preventative therapy, to patients in need thereof. The present invention fills these needs.